1. Field of the Invention
This invention relates to a novel Bacillus subtilis strain useful as the host for the highly efficient secretory production of desired proteins by causing the host to secrete the desired proteins using recombinant DNA techniques and to a method for the highly efficient secretory production of the desired proteins by using the strain as the host.
More particularly, this invention relates to a method for producing the desired protein efficiently by utilizing a combination of (a) a novel B. subtilis strain as the host obtained by introducing a gene for the stimulation of extracellular protease levels derived from a bacterium of the genus Bacillus into a recipient B. subtilis strain whose extracellular protease activities are already reduced so that the extracellular protease activities of the resultant strain become lower than those of the recipient strain; and (b) a recombinant plasmid containing a gene coding for the desired protein joined to an extracellular protease gene or a DNA sequence derived therefrom so as to express the gene and secrete the protein thus expressed.
2. Description of the Prior Art
Recombinant DNA techniques have recently made it possible to cause microorganisms as the host to express foreign genes and therefore desired foreign gene products can be produced by using these techniques.
As the host microorganism, various bacteria including Escherichia coli, B. subtilis and various yeasts have been generally used.
From the viewpoint of efficient material production, attempts to secrete foreign proteins into the culture media of transformants capable of producing the foreign proteins have been made. As a host microorganism useful for this purpose, the genus Bacillus having the character of secretion of large amounts of extracellular proteins such as protease, amylase and others has been primarily regarded as a suitable host.
Among the genus Bacillus, B. subtilis has in particular attracted attention, not only because it is safe, but also because much information about it has been accumulated in the fields of genetics, biochemistry, molecular biology and applied microbiology. Therefore, efforts have been made to construct hostrecombinant plasmid systems capable of secreting foreign proteins in the culture media by using B. subtilis as the host.
In many cases in the attempts in the prior art to cause B. subtilis as the host to secrete foreign proteins, the accumulation levels of the secreted proteins were however still low, particularly where the foreign proteins derived from higher eukaryotes. Therefore, the techniques for the secretion of foreign proteins in the prior art are not yet satisfactory in terms of industrial production.
It has been presumed that small accumulation of the secreted proteins may be caused by the degradation of the foreign proteins derived from higher eukaryotes during or after secretion, because, although the mRNAs were synthesized in sufficiently large amounts, the foreign proteins were accumulated only in small amounts (Reference 1).
The B. subtilis strain whose extracellular protease activities are reduced to avoid the degradation of the desired protein has been regarded as a suitable host in view of the existing state of the art.
However, no suitable B. subtilis strains as the host for the secretion of foreign proteins originating from higher eukaryotes in sufficient amounts are known from various experimental results reported in the prior art.
For example, Palva et al. constructed a B. subtilis strain whose extracellular protease activities were reduced, introduced into it a recombinant plasmid containing a combination of an .alpha.-amylase signal peptide coding DNA sequence and a human interferon-.alpha. gene joined to the downstream side thereof and then obtained transformants. In their experiment on the secretion of human interferon-.alpha. by the transformants, the accumulation levels reached 10.sup.5 units per 1 l of the culture medium. This value corresponds to approximately 1 mg/l-culture medium of human interferon-.alpha., and this level is much lower than that of the .alpha.-amylase (Reference 2).
The present inventors attempted the secretory production of human interferon-.beta. by using a B. subtilis strain whose extracellular neutral and alkaline protease activities were reduced as the host. In this case, although the strain whose protease activities were reduced was used as the host, the accumulation level (10 mg/l-culture medium) of the secreted human interferon-.beta. was not as high as the present inventors had expected. They also confirmed that the foreign protein produced was rapidly degraded in the absence of a protease inhibitor in the culture medium (Reference 3).
As described above, it has ben difficult in the prior art methods to accumulate the desired foreign proteins derived from higher eukaryotes in large amounts in the culture supernatants only by using as the host B. subtilis strains whose extracellular neutral and alkaline protease activities were reduced by the prior art methods.